Advances in the field of recombinant DNA technology have resulted in the cloning of various naturally occurring DNA sequences and the expression of the underlying recombinant DNA to produce biologically active recombinant polypeptides. For example, human growth hormone has been produced in E. coli by fusing the coating sequences for this protein to an E. coli promoter (1). In a second example, tissue plasminogen activity, another rare human protein, has also been produced in E. coli (2).
Modifications of certain recombinant polypeptides have been made to investigate the properties of such modified polypeptides. To this end, naturally occurring DNA sequences have been cloned and modified by deleting or replacing amino acid residues of the naturally occurring polypeptide to modify the physical properties of the recombinant polypeptide, as for example, by site directed mutagenesis as disclosed in U.S. Pat. No. 4,518,584.
Recombinant polypeptides have also been modified by fusing recombinant DNA sequences. For example, the signal sequence from a plasmid-derived beta-lactamase was positioned at the amino-terminus of proinsulin through a common restriction site to facilitate the secretion of proinsulin (3).
Two different human alpha interferon DNA sequences have been combined by way of a common restriction site to form a DNA sequence containing sequences from alpha-1 interferon and alpha-2 interferon as described by Weissman (4). The alpha interferons expressed by such fused alpha interferon DNA sequence, however, demonstrated limited biological activity.
A major limitation in producing modified polypeptides by combining the underlying DNA at a restriction site, at more than one restriction site by way of a bridging synthetic oligonucleotide or by combining synthetic oligonucleotides to form the entire modified DNA sequence lies in the enormous amount of work which is required to produce a particular modified recombinant polypeptide. For example, such modifications require knowledge of the DNA and/or polypeptide sequence which if not available must be determined. Moreover, even if such sequences are known, the task of producing a modified polypeptide is far from simple and may result in a biologically inactive molecule.
Weber, et al., (5) disclose a method for making modified genes by in vivo recombination between DNA sequences encoding an alpha-1 and an alpha-2 human interferon sequence. A linear DNA sequence containing a plasmid vector flanked by the alpha-2 interferon gene on the 5' end and a portion of alpha-1 interferon gene on the 3' end was used to transfect a rec A positive strain of E. coli. Circularization of the linear plasmid by in vivo recombination between the partially homologous interferon gene sequences produced a number of modified interferon genes containing various portions of the alpha-1 and alpha-2 interferon gene sequences. Weber reports that some of these modified alpha interferon genes expressed modified alpha interferons having biological activity similar to unmodified alpha-2 interferons.
The efficiency of producing modified genes and polypeptides by in vivo circularization (recombination) of linear plasmids, as disclosed by Weber (5), is limited by the relative inefficiency of linear plasmids to transfect microorganisms as compared to circular plasmids. In addition, the two different but related genes on such linear plasmids are always separated by a replicable plasmid sequence. Circularization requires that the ends of the vector containing the two genes overlap to bring the two genes into close proximity with each other. The efficiency of recombination may therefore be limited by the linearity of such plasmid constructions.
Accordingly, it is an object herein to provide circular vectors containing replicable DNA sequences and DNA sequences encoding at least two different parental polypeptides and recombined circular vectors containing said replicable DNA sequences and a hybrid DNA sequence encoding a hybrid polypeptide corresponding to part of each parental polypeptide.
It is a further object of the present invention to provide efficient processes for making such recombined circular vectors and hybrid polypeptides.
Further, an object of the invention is to provide additional processes for isolating such recombined circular vectors.
Further, an object of the present invention is to provide biologically active hybrid polypeptides containing segments of polypeptide sequences derived from at least two parental polypeptides.
Still further, an object of the present invention is to provide biologically active hybrid enzymes such as hybrid amylases and hybrid proteases.